EP4532686A1 - Procédés de fabrication de cellules progénitrices d'oligodendrocytes - Google Patents

Procédés de fabrication de cellules progénitrices d'oligodendrocytes

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Publication number
EP4532686A1
EP4532686A1 EP23812323.6A EP23812323A EP4532686A1 EP 4532686 A1 EP4532686 A1 EP 4532686A1 EP 23812323 A EP23812323 A EP 23812323A EP 4532686 A1 EP4532686 A1 EP 4532686A1
Authority
EP
European Patent Office
Prior art keywords
opcs
progenitor cells
cells
oligodendrocyte progenitor
npcs
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP23812323.6A
Other languages
German (de)
English (en)
Inventor
Lev STARIKOV
Juliana HSU
Thomas CUTIA
Mark Ebel
Mark Tomishima
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
BlueRock Therapeutics LP
Original Assignee
BlueRock Therapeutics LP
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by BlueRock Therapeutics LP filed Critical BlueRock Therapeutics LP
Publication of EP4532686A1 publication Critical patent/EP4532686A1/fr
Pending legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0618—Cells of the nervous system
    • C12N5/0622—Glial cells, e.g. astrocytes, oligodendrocytes; Schwann cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/08—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from cells of the nervous system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells

Definitions

  • FIG. 3A-3B (A) Suspension cells were cultured either in stationary vessels or agitation format in a bioreactor. (B) Agitation generated more uniform sphere size and shape. Flow analysis showed SOX9+/NKX2-2+ double expression. Immunofluorescence staining of sphere sections at Day 20 (D20) showed differences in sphere organization shown by SOX9 and NKX2.2 expression. Spheres dissociated at Day 60 (D60) are stained for 04 by flow and IF. Stationary and agitation spheres showed similar gene expression patterns for neural and glial progenitor markers as assayed by RT-qPCR panel. Scale bars, 200pm in bright field images and 100pm in fluorescent images.
  • FIG. 4A-4B The % of OPCs committed to oligodendrocyte fate at Day 60 is regulated by WNT (Wingless/Integrated) signaling.
  • WNT Wired/Integrated
  • the co-expression of SOX10 and 04 antigen signify the fate commitment of OPC to oligodendrocyte lineage.
  • the percentages of SOX10 and 04 positive cells at Day 60 increase to >50% with the addition of WNT modulator between Day 40 to Day 60 (B) compared to those without any manipulation (A).
  • WNT modulator (agonist) added at 3uM.
  • FIG.5. Dissociation and cry opreservation of oligodendrocyte progenitor cell (OPC) cell product.
  • Spheres were enzymatically dissociated into a single cell suspension by passing through a 70pm strainer. Dissociation progress was monitored by tracking cell concentration over time, cell health was simultaneously monitored by lactate dehydrogenase (LDH) rel as an indicator of cell death. Data shown are technical replicates from one representative experiment. Single cells were cryopreserved using a control rate freezing program and transferred to liquid nitrogen long term storage. Agitation bioprocess supports higher seeding density than stationary culture and translates to significantly higher cell yield by Day 60. Upon thaw, oligodendrocyte progenitor cell (OPC) cell product was comparable between both stationary and agitation workflows. Individual data points in bar graphs represent 2-3 separate experiments with 2-3 technical replicates per experiment.
  • FIG. 6 Comparison of the single cell transcriptome of stationary and agitation spheres at Day 20 of culture. Unbiased single-cell RNA-sequencing showed that both culture methods induce NKX2-2 ventral fated neuronal (MNX1) and glial progenitor (SOX9) populations.
  • MNX1 ventral fated neuronal
  • SOX9 glial progenitor
  • FIGS. 7A-7C Day 60 dissociated cells from stationary culture were transplanted into Pl 3 MBP (myelin basic protein) deficient shiverer mice (Rag2/112 KO) in an immune- compromised genetic background.
  • Pl 3 MBP myelin basic protein
  • Rag2/112 KO myelin basic protein
  • FIGS. 7A-7C Day 60 dissociated cells from stationary culture were transplanted into Pl 3 MBP (myelin basic protein) deficient shiverer mice (Rag2/112 KO) in an immune- compromised genetic background.
  • B Schematic of OPC injection.
  • C Representative images of OPCs integrated in the corpus callosum (left) and cerebellum (right). Human cells express STEM121 (red), and mature oligodendrocytes restore the expression of the myelin protein MBP (green). Scale bars are 200pm.
  • FIG. 8 Comparison of the effect of OPCs vs. vehicle in ataxic mice. OPCs rescued ataxic gait compared to vehicle controls.
  • the term “subject” refers to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, rodents, and the like, which is to be the recipient of a particular treatment.
  • the terms “subject” and “patient” are used interchangeably herein in reference to a human subject.
  • non-human animals refers to all non-human animals including, but not limited to, vertebrates such as rodents, non-human primates, ovines, bovines, ruminants, lagomorphs, porcines, caprines, equines, canines, felines, aves, etc.
  • cell culture refers to any in vitro culture of cells. Included within this term are continuous cell lines (e.g., with an immortal phenotype), primary cell cultures, transformed cell lines, finite cell lines (e.g., non-transformed cells), and any other cell population maintained in vitro.
  • in vitro refers to an artificial environment and to processes or reactions that occur within an artificial environment.
  • In vitro environments can consist of, but are not limited to, test tubes and cell culture.
  • in vivo refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.
  • PSCs pluripotent stem cells
  • ESCs embryonic stem cells
  • iPS cells induced pluripotent stem cells
  • ES cells and iPS cells have their usual meaning in the art.
  • Terms such as “treating” or “treatment” or “to treat,” as used herein, refer to therapeutic measures that cure, restore regenerative function, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic disease or disorder. Thus, those in need of treatment include those already with the disorder.
  • a subject is successfully “treated” for a disease or disorder if the subject shows, e.g., total, partial, permanent, or transient, alleviation or elimination of any symptom associated with the disease or disorder.
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • exemplary routes of administration for the oligodendrocyte progenitor cells (OPCs) prepared by the methods disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • phrase parenteral routes of administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrastemal injection and infusion, as well as in vivo electroporation.
  • NPCs Neural Progenitor Cells
  • the first phase of the disclosed process is to derive neural progenitor cells (NPCs) from pluripotent stem cells by a dual-SMAD inhibition method (Chambers S.M, Craft C.A, Papapetrou E.P, Tomishima M, Sadelain M, Studer L. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 2009;27(3):275-280. Epub 2009 Mar 1. Erratum in: Nat Biotechnol. 2009 May,27(5):485.) which is a well-established method to derive neural progenitor cells fro pluripotent stem cells.
  • NPCs neural progenitor cells
  • the present disclosure describes a dissociation procedure which is scalable, time efficient with reduced operator handling and has improved batch-to-batch consistency. [0057] The dissociation procedure combines enzymatic treatment and mechanical agitation. Additionally, the current method eliminates the need for exogenous DNase I during enzymatic treatment. Collectively, the current method results in high viable cell yield and post-thaw cell viability exceeding the benchmark of 70%.
  • agitators were tested, including but not limited to DASbox mini-bioreactor, Thermo Mixer C (Eppendorf) or Octo Dissociator (Miltenyi). All three provided tunability for rotational speed and heating (37°C) capability.
  • improved scalability relies on two components, source of enzymes and vessels used to perform the dissociation and both components needed to be scalable for this procedure to work.
  • different types of enzyme mixtures/dissociation agents were employed including but not limited to Miltenyi Neurosphere Dissociation Kit (P), Accutase (lx), AccuMax, lOx TrypLE Select. Accutase followed by 2xTrypLE Select, IxAccuMax plus 2xTrypLE Select or 4x TrypLE Select diluted in HBSS (Hanks' Balanced Salt Solution).
  • the enzymatic treatment comprises lx AccuMax and 2x TrypLE Select diluted in HBSS or 4x TrypLE Select diluted in HBSS. The efficiency of enzymatic dissociation depends on the sufficient enzyme to cell mass ratio.
  • the enzyme to cell mass ratio is based on volume.
  • a sufficient ratio of cell (spheres): enzyme is 1:20 to 1: 100.
  • the ratio is 1 :25 to 1:90, 1 :30 to 1 :80, 1 :40 to 1 :70, 1 :50 to 1:60.
  • the DASbox minibioreactor allows enzyme supplies at scalable quantities. The DASbox mini-bioreactors hold up to 250 mL solution in each unit.
  • the dissociation method using DASbox mini-bioreactors affords a significantly shortened time course.
  • the mini-bioreactors of the disclosure enables a 3-hour procedure that can process up to about 800 mL or more of oligosphere culture.
  • the cell yield is defined as the number of viable single cells per milliliter of the original culture volume.
  • the cell yield using DASbox mini-bioreactors in accordance with the present disclosure is about 1.3 millions/mL, about 1.35 millions/mL, about 1.4 millions/mL, about 1.45 millions/mL, about 1.5 millions/mL, about 1.55 millions/mL, about 1.59 millions/mL, about 1.60 millions/mL, about 1.65 millions/mL or about 1.69 millions/mL.
  • the enzymatic treatment does not require addition of exogenous DNase I into the dissociation mixture.
  • exogenous DNase I Conventionally, an issue upon dissociating any type of spheroids is the release of genomic DNA into the dissociation mixture, due to various degrees of compromised cell membrane or cell death in the process. As a result, the viscosity of the dissociating mixture is increased, and cell clumps may occur.
  • Adding exogenous DNase I into the dissociation mixture is a common, art- recognized mitigation measure.
  • the use of DNase I introduces foreign biological matter into the cell product. Complete elimination of DNase I after the procedure is either by dilution via extensive washing, which increases operator handling, or heat-inactivation at 65°C, which is incompatible with most cells. The method disclosed herein, has completely eliminated the need for DNase I.
  • a lactate dehydrogenase release serves as a cell health indicator during sphere dissociation.
  • Cry opreservation is a process that maintains biological samples in a state of suspended animation at cryogenic temperature for any considerable period and is used to preserve the fine structure of cells.
  • the freezing behavior of the cells can be altered in the presence of a cryoprotective agent (also called cryoprotectant), which affects the rates of water transport, nucleation, and ice crystal growth.
  • a cryoprotective agent also called cryoprotectant
  • the single oligodendrocyte progenitor cells were cryopreserved using a freezing process (control rate freezer) that is step-wise.
  • the use of CRF ensures a batch-to-batch reproducibility.
  • the type of cryoprotectant does not play an important role in and of itself.
  • oligodendrocyte progenitor cells are generated from induced pluripotent stem cells, derived from a healthy subject. PSC-derived oligodendrocyte progenitor cells (OPCs) are administered into the central nervous system of a subject to be treated.
  • FIG. 7A- 7C show that, in accordance with the present disclosure, the PSC-derived OPCs successfully engrafted and matured into myelin basic protein (MBP) -expressing oligodendrocytes in the hypomyelinated mouse brain. Significantly, the mature oligodendrocytes successfully restored the expression of the myelin basic protein (MBP).
  • MBP myelin basic protein
  • Neural progenitor cells were differentiated in a suspension culture in an impeller-driven DASbox mini -bioreactor for 60 days to generate oligospheres. The oligospheres were then settled for 30 minutes. The suspension culture medium was pumped out and dissociation reagent/enzyme mix (lx AccuMax and 2x TrypLE Select diluted in HBSS (Hanks' Balanced Salt Solution) or 4x TrypLE Select diluted in HBSS) was added. The dissociation reagent/enzyme mix did not include exogenous DNase I. The final bioreactor volume was 200mL.
  • the impeller of the DASbox mini-bioreactor was turned on to 100 rpm for 1 hour at 37°C. After 1 hour, the impeller speed was increased to 400 rpm for 2 hours at 37°C. ImL samples were taken every 30 minutes for cell counts and metabolite measurements. After 3 hours, the impeller was stopped when the oligospheres were dissociated into single oligodendrocyte progenitor cells (OPCs). Then the entire 200 mL cell suspension volume was taken out and passed through a 70pm strainer and into a 250mL centrifuge tube. Then it was centrifuged for 200g x lOmin.
  • OPCs oligodendrocyte progenitor cells
  • oligodendrocyte progenitor cells were dissociated into single cells, they were immediately resuspended into cryoprotectant solution, aliquoted, and slowly frozen to be stored in liquid nitrogen. After average cell count was recorded as described in Example 1, the cells were centrifuged for 200g x lOmin and resuspended in cryoprotectant solution (StemCell Banker) at either 5 million or 10 million/mL pre-chilled and labeled IrnL cryotubes. These cryotubes/aliquots were brought on ice to control rate freezer (CRF) and the program was started. After the program was complete, the aliquots were transferred immediately to dry ice and then into liquid nitrogen tank storage.
  • OPCs oligodendrocyte progenitor cells
  • the CRF freezing program is a controlled, step-wise freezing process, designed specifically for mammalian cells, and batch-to-batch reproducibility. This program designs six steps to run samples from room temperature to -130°C. In some embodiments samples run from room temperature to -125°C. Tn some embodiments samples run from room temperature to - 120°C. Tn some embodiments samples run from room temperature to -115°C. In some embodiments samples run from room temperature to -110°C. In some embodiments samples run from room temperature to -105°C. In some embodiments samples run from room temperature to -100°C. In some embodiments samples run from room temperature to -95°C. In some embodiments samples run from room temperature to -90°C.
  • samples run from room temperature to -40°C. In some embodiments samples run from room temperature to -15°C. In some embodiments samples run from room temperature to -12°C. In some embodiments samples run from room temperature to -10°C.
  • a key component is the first 10 minutes of rapid cooling to 0°C. This ensures every batch of cells reaches 0°C within a tightly-controlled timeframe, and therefore, sample temperatures are synchronized before the next cooling phase begins.
  • OPCs The efficacy of OPCs was assessed in the Shiverer mouse, a hypomyelinated mouse model. OPCs were transplanted into the brainstem and cerebellum (regions with large volumes of white matter, and importance for motor coordination) of Shiverer mice. OPCs rescued ataxic gait compared to vehicle controls (FIG. 8), a key phenotype observed in clinical populations. Furthermore, rescue in ataxic gait correlated with functional remyelination of the cerebellum as measured by the presence myelin basic protein (MBP) expression that is absent in the Shiverer mouse (data not shown). Conduction velocity was measured across major axon bundles, as a readout for functional myelination. Taken together, these data demonstrated that OPCs are sufficient to rescue molecular, functional, and subsequent behavioral phenotypes in a hypomyelinated mouse, suggesting that OPCs provide a treatment option for demyelinating disorders where existing therapeutics are limited.
  • MBP myelin

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Abstract

La présente invention concerne des procédés de production de cellules progénitrices d'oligodendrocytes (OPC). L'invention concerne en outre des méthodes de traitement de maladies démyélinisantes à l'aide de cellules progénitrices d'oligodendrocytes (OPC).
EP23812323.6A 2022-05-24 2023-05-04 Procédés de fabrication de cellules progénitrices d'oligodendrocytes Pending EP4532686A1 (fr)

Applications Claiming Priority (3)

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US202263345251P 2022-05-24 2022-05-24
US202263356536P 2022-06-29 2022-06-29
PCT/US2023/020916 WO2023229819A1 (fr) 2022-05-24 2023-05-04 Procédés de fabrication de cellules progénitrices d'oligodendrocytes

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EP4532686A1 true EP4532686A1 (fr) 2025-04-09

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US (1) US20250333694A1 (fr)
EP (1) EP4532686A1 (fr)
JP (1) JP2025516957A (fr)
KR (1) KR20250017234A (fr)
CN (1) CN119278260A (fr)
AU (1) AU2023276337A1 (fr)
CA (1) CA3256595A1 (fr)
WO (1) WO2023229819A1 (fr)

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WO2012032521A2 (fr) * 2010-09-07 2012-03-15 Technion Research & Development Foundation Ltd. Nouveaux procédés et milieux de culture destinés à la culture de cellules souches pluripotentes
RU2687379C2 (ru) * 2012-12-31 2019-05-13 Янссен Байотек, Инк. Суспендирование и кластеризация плюрипотентных клеток человека с целью их дифференцировки в панкреатические эндокринные клетки
WO2014124087A1 (fr) * 2013-02-06 2014-08-14 University Of Rochester Cellules progénitrices d'oligodendrocyte issues de cellules pluripotentes induites pour le traitement de troubles de la myéline
ES2527796B1 (es) * 2013-07-29 2015-11-10 Universidad De Almería Método para la cuantificación fluorimétrica de la enzina LDH en disolución
CA3061165A1 (fr) * 2017-04-26 2018-11-01 Memorial Sloan-Kettering Cancer Center Cellules cryoconservees pretes a l'emploi
IL281628B2 (en) * 2018-09-19 2025-06-01 Lineage Cell Therapeutics Inc Methods for differentiating pluripotent stem cells in dynamic suspension culture
US11603518B2 (en) * 2019-01-23 2023-03-14 Asterias Biotherapeutics, Inc. Dorsally-derived oligodendrocyte progenitor cells from human pluripotent stem cells
WO2020219696A1 (fr) * 2019-04-26 2020-10-29 The Regents Of The University Of California Dispositifs et procédés de génération de cellules progénitrices d'oligodendrocytes
CA3152504A1 (fr) * 2019-09-06 2021-03-11 Keio University Procede de production d'agregat cellulaire comprenant des cellules progenitrices gliales

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US20250333694A1 (en) 2025-10-30
CA3256595A1 (fr) 2023-11-30
CN119278260A (zh) 2025-01-07
JP2025516957A (ja) 2025-05-30
AU2023276337A1 (en) 2024-12-05
KR20250017234A (ko) 2025-02-04
WO2023229819A1 (fr) 2023-11-30

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